The long-range goal of these studies is to understand how the cytoplasmic protein ZO-1 organizes proteins at tight junction (TJ) in order to create a barrier in the paracellular pathway. ZO-1 is a member of the multi-domain MAGUK protein family (membrane-associated guanylate kinase). It binds to 3 different transmembrane proteins and at least 10 cytoplasmic proteins and actin. There is strong evidence that ZO-1 is a critical scaffold for assembling the TJ but little information about what regulates the interaction between ZO-I and its binding partners. The binding activity of other MAGUK proteins is regulated by intra- and intermolecular interactions between specific functional and structural domains. Our preliminary studies show the GUK domain of ZO-1 binds occludin, cingulin and alpha-catenin, while an adjacent acidic domain can inhibit these interacts. In Drosophila ZO-1 the acidic domain is alternatively spliced and correlates with different subcellular locations and rescue of different signaling pathways in the ZO-1 null mutant. In mammalian cultured cells, expression of ZO-1 lacking the acidic domain induces structurally aberrant ectopic TJs on the apical and lateral cell surfaces, implying the GUK-acid interaction controls a step in assembly. As junctions assemble in cultured cells the WT protein temporally precedes ZO-1 lacking the acid domain, suggesting the domain is required to respond to kinetic assembly signals. We will characterize the mechanisms by which the acidic domain regulates ligand binding to the GUK domain of ZO-1 and the functional consequences for TJ structure and function. We will characterize these protein interactions using yeast 2-hybrid and fusion protein assays and truncated fragments in cultured epithelial cells. Isothermal titration calorimetry will be used to determine the affinity of the GUK domain and its protein ligands, their regulation by the acidic domain and possible regulation by protein phosphorylation. The atomic structures and their mechanisms of regulated interaction will be visualized by solving the x-ray crystallographic structures of occludin bound to the GUK domain with and without the SH3 and acidic domains, and with and without phosphorylation. Similar studies will be performed on alpha-catenin and cingulin as time permits. These studies are the first to describe the structural basis for regulated assembly of tight junctions and will provide insight into the mechanisms controlling assembly.